Cardiac regulatory protein will be isolated from experimentally-induced ischemic hearts. Extraction, which will be carried out at low ionic strength using purified myofibrils, does not involve organic solvent treatment. Endogenous protein kinase associated with regulatory proteins isolated from the ischemic myocardium will be characterized. A comparison will be made with counterparts isolated from control myocardium. Control and ischemic regulatory proteins will be fractionated into their components (troponin, tropomyosin and bound protein kinase) using hydroxylapatite column chromatography. Troponin isolated from control and ischemic myocardium will be fractionated into its components (troponin-T, troponin-I, and troponin-C) using DEAE-cellulose (DE-52) in the presence of 6 M urea. Separation will be carried out with a linear NaCl gradient. Phosphorylation of the above protein fractions will be examined using bound protein kinase, phosphorylase-b kinase and cytoplasmic enzyme. The properties of the phosphorylated proteins of normal and ischemic myocardium, with respect to their calcium binding and their ability to modify actomyosin ATPase, will be studied. Myosin, actin and troponin-I will be isolated from the control and ischemic myocardium using affinity column chromatography. This technology has several advantages over existing methods and is useful in instances where the availability of tissue is limited, such as in ischemic and biopsy samples. Phosphatases will be isolated from the cytosol fraction of the myocardium and from natural actomyosin preparations. Dephosphorylation of phosphorylated of myofibrillar proteins will be carried out. A possible defect in the bound protein kinase and cyclic AMP-dependent phosphorylation myofibrillar proteins isolated from the ischemic myocardium will be examined.